Abstract

It is known that binding sites with endothelinA(ET)A and ETB receptor characteristics coexist in human heart but little is known about the receptors that mediate cardiostimulant effects of ET receptor agonists or their consequences. Functional studies were performed on isolated human cardiac tissues. The maximal positive inotropic effects of ET-1 were right atrium > left atrium = right ventricle. The rank order of potencies of agonists in right atrium was sarafotoxin S6c > ET-1 = ET-2 ≥ ET-3. The ETA receptor-selective compounds BQ123 (10 μM) and A-127722 (1 μM) only slightly blocked (<0.5 log-unit shift) the effects of lower concentrations of ET-1, and the ETB receptor antagonist Ro46-8443 (10 μM) did not cause blockade. SB 209670 caused concentration-dependent rightward shifts of ET-1 and sarafotoxin S6c concentration-effect curves with Schild slopes not different from one and affinities (−logM KB) of 7.0 and 7.9, respectively. ET-1 caused arrhythmic contractions in right atrial trabeculae that were prevented by 10 μM SB 209670 but not 10 μM BQ123 or 1 μM A-127722, precluding ETA receptors. ET-1 caused a higher incidence of arrhythmic contractions in tissues taken from patients treated with β-blockers before surgery than in tissues from non-β blocker-treated patients. Sarafotoxin S6c produced arrhythmias that were prevented by SB 209670. The positive inotropic effects of ET-1 in human right atrial myocardium are mediated mostly by a non-ETA, non-ETB receptor. Ventricular inotropic ET receptors differ from atrial inotropic ET receptors. ET-1 induced arrhythmic contractions in human atria do not appear to be mediated by an ETA receptor.

Early studies showed that endothelin-1 (ET-1) can directly affect the contractile state of cardiac tissues. Positive inotropic effects to ET-1 have consistently been observed in atrial tissues from many species, including humans (Davenport et al., 1989; Moravec et al., 1989; Meyer et al., 1996), that were greater than those observed in corresponding ventricular tissues, including those from humans (Davenport et al., 1989; Moravec et al., 1989). It is generally thought that ET-1 combines with specific cell surface endothelin receptors that mediate its effects. Radioligand binding, quantitative receptor autoradiography, polymerase chain reaction, and in situ hybridization studies showed the presence of two receptor subtypes, ETA and ETB, in human cardiac tissues (Bax et al., 1993; Molenaar et al., 1993). Since then, there have been increasing numbers of reports of ET receptors that do not fit the ETA or ETBreceptor classification in a variety of tissues (Bax and Saxena, 1994). Using a single concentration of the ETA selective antagonist BQ123 [cyclo(d-Trp-d-Asp-Pro-d-Val-Leu)] (200 nM), Meyer et al. (1996) suggested that ETAreceptors mediated the inotropic effects of ET-1 in human right atrium. The aim of our study was to use a wider range of ET receptor agonists and antagonists to more fully characterize the receptors that are responsible for the inotropic effects of agonists in human right atrium. We were interested to know whether in addition to the reported ETA cardiac receptor (Meyer et al., 1996), ETB and/or non-ETA/ETB receptors were also responsible for the cardiostimulant effects of ET receptor agonists.

There is evidence for a direct arrhythmogenic effect of ET-1. Arrhythmias following ischemia-reperfusion in rat hearts have been shown to be caused by ET-1 (Brunner and Kukovetz, 1996) and procedures that lower ET-1 levels (angiotensin-converting enzyme inhibition, bradykinin) or block the ET-1 receptor {SB 209670 [(+)-(1S,2R,3S)-5-propoxy-1-(3,4-methylenedioxyphenyl)-3-(2-carboxymethoxy-4-methoxyphenyl)indane-2-carboxylic acid disodium)]} prevent ischemia-reperfusion arrhythmias (Brunner and Kukovetz, 1996). In AT-1 cells, an atrial tumor myocyte cell line derived from transgenic mice, ET-1 caused the appearance of spontaneous diastolic calcium oscillations in both electrically driven and quiescent cells (Jiang et al., 1996). We were interested to know whether ET-1 was arrhythmogenic in human atrial tissue. For this purpose, we used a model previously described by Kaumann and colleagues (Kaumann and Sanders, 1993, 1994; Sanders et al., 1996) in human right atrial tissue in which it was shown that stimulation of Gs protein-coupled receptors, β1- and β2-adrenoceptors, 5-hydroxytryptamine (5-HT)4- and H2-receptors cause pacing frequency-dependent arrhythmic contractions.

Patients undergoing coronary artery bypass grafting, aortic valve replacement, or the combined procedures were excluded from comparison with right atrial tissue from terminal heart failure tissues if patients had congestive cardiac failure. Patients were diagnosed as having congestive cardiac failure if on preoperative clinical assessment they had symptoms, signs, and medical treatment consistent with the diagnosis and an ejection fraction <30%. Ejection fraction was determined via echocardiography or left ventriculogram. Clinical features included exertional dyspnoea, orthopnea, basal crepitations, and medical management with diuretics or angiotensin-converting enzyme inhibitors. Information was obtained prospectively and recorded at the time of operation by the anesthesiologist. One patient with an ejection fraction <30% was excluded retrospectively based on clinical assessment.

These studies were approved by the ethics committees of the Royal Melbourne Public and Private Hospitals (BOMR 10/94), The Alfred Hospital, Prahran (33/89), The University of Melbourne (HREC 951686), The Prince Charles Hospital (EC9876), and The University of Queensland (H/29/Med/PCH/NHMRC/99).

For procedures from which right atrial appendages were obtained, premedication usually included 150 mg of ranitidine orally on the night before surgery and 150 mg of ranitidine, 15/0.3 to 20/0.4 mg s.c. papaveretum/scopolamine, 5000 I.U. s.c. heparin, and 5 to 10 mg diazepam orally ∼2 h before surgery. Anesthesia was induced with 20 μg/kg fentanyl supplemented with midazolam, propofol, or isoflurane. For some experiments, patients were subdivided into two groups according to whether they were treated chronically with selective β1-adrenoceptor antagonists or not before surgery. Those receiving β1-adrenoceptor antagonists were treated with either atenolol (25–50 mg daily) or metoprolol (50–100 mg daily). Patients who were receiving antiasthma medication were not prescribed β-adrenoceptor antagonists; however, drug therapy for both groups included the use of hypolipidemics, hypoglycemics, diuretics, angiotensin-converting enzyme inhibitors, nitrates, and calcium antagonists.

For cardiac transplantation, premedication consisted of temazepam (10–20 mg) or midazolam (2–4 mg). Anesthesia was induced with a combination of propofol infusion and fentanyl bolus (10 μg · kg−1) supplemented with midazolam. Maintenance was achieved either with isoflurane vapor or with propofol infusion, augmented by fentanyl and midazolam boluses. Table1 provides a summary of patient age, sex, surgical procedure, and drug administration before surgery.

Effects on Human Atrial and Ventricular Contractile Force.

Tissues were incubated with 300 nM CGP 20712A [2-hydroxy-5(2-((2-hydroxy-3-(4-((1-methyl-4-trifluoromethyl) 1H-imidazole-2-yl) -phenoxy) propyl) amino) ethoxy)-benzamide monomethane sulfonate] and 50 nM ICI 118,551 [erythro-d,l-1(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol] for at least 60 min to block β1- and β2-adrenoceptors, respectively. In some experiments, ET receptor antagonists also were added and equilibrated with atrial tissues from nonfailing hearts for at least 60 min. Cumulative concentration-effect curves to ET receptor agonists in the absence or presence of antagonists were determined by sequential administration of agonist to the tissue bath in amounts that increased the total concentration by ½ log unit.

To determine whether ET-1 and sarafotoxin S6c caused positive inotropic effects by stimulation of the same receptor, concentration-effect curves were established to ET-1 in the absence or presence of sarafotoxin S6c. In these experiments sarafotoxin S6c was added cumulatively in ½ log units commencing at 200 pM to a final concentration of 200 nM and in one additional experiment to 1 μM. Concentration-effect curves were completed by raising the Ca2+ concentration to 9.25 mM.

Human Coronary Arteries.

After surgical removal of the heart from one patient with idiopathic dilated cardiomyopathy, large human epicardial coronary arteries were dissected, placed immediately into ice-cold preoxygenated modified Krebs' solution (described above), transported to the laboratory, cleared of fat and connective tissue, and set up in the organ bath as described in Kaumann et al. (1994). Briefly, the endothelium was removed by gently rubbing the lumen with paper towel. Helicoidal strips were mounted in the same apparatus used for cardiac muscle and resting force was adjusted to ∼30 mN at the beginning of the experiment. The incubation medium was exchanged as described above. Tissues were allowed to stabilize for 3 h before addition of 90 mM KCl followed by washout and readdition of KCl 2 h later. Following washout, 10 μM BQ123 or 1 μM A-127722 [trans-trans-2-(4-methoxyphenyl)-4-(1,3-benzodioxol-5-yl)-1((N,N-dibutylamino)carbonylmethyl)pyrolidine-3-carboxylic acid] was added to some organ baths and incubated for 2 h before commencement of a cumulative concentration-effect curve to ET-1.

Arrhythmia Studies.

The ability of ET-1 and sarafotoxin S6c to cause arrhythmic contractions in human right atrium was determined in a staircase model as described in detail for stimulation of human atrial β1- and β2-adrenoceptors (Kaumann and Sanders, 1993), 5-HT4 receptors (Kaumann and Sanders, 1994), and H2-receptors (Sanders et al., 1996). Briefly, atrial tissues of nonfailing hearts from patients undergoing coronary artery bypass grafting, aortic valve replacement or a combination of both procedures were set up as described above for the determination of cumulative concentration-effect curves and incubated with 300 nM CGP 20712A and 50 nM ICI 118,551 to block β1- and β2-adrenoceptors with or without ET receptor antagonists for at least 60 min. The pacing frequency was then set at 0.1 Hz and reset at 0.2, 0.5, 1, and 2 Hz at 2-min intervals (forward staircase). The staircase was then run backward (2–0.1 Hz), with 2-min intervals during which the stimulator was turned off (rest periods) between each 2-min stimulation period. The pacing rate was then set at 1 Hz and on stabilization, ET-1 or sarafotoxin S6c was added to the tissue bath. After equilibration, the backward staircase (2–0.1 Hz) was established with 2-min rest periods between each frequency repeated in the presence of ET-1 or sarafotoxin S6c.

To determine whether spontaneous contractions could be evoked by ET-1 in nonpaced tissues, right atrial tissues from patients undergoing coronary artery bypass grafting were first set up as described above for the determination of cumulative concentration-effect curves. Tissues were paced at 1 Hz, the length set at 50% Lmax and incubated with 300 nM CGP 20712A and 50 nM ICI 118,551 for 50 min. The stimulator was then turned off and tissues were exposed to 100 nM ET-1; other tissues served as controls. If spontaneous contractions were observed, 100 nM verapamil was added to some tissues with other tissues serving as time controls.

Experimental Design and Analysis.

Changes in contractile force above basal were calculated. Where two or more strips from one patient were used, mean changes in contractile force above basal values were calculated for each concentration. For agonists, pEC50 (−log concentration causing 50% of the maximal response) and maximal responses, expressed as a percentage of the response to 9.25 mM Ca2+, were measured.

pEC50 and maximal response values for ET-1 or sarafotoxin S6c in the presence of increasing concentrations of SB 209670 were obtained. Schild-plots (Arunlakshana and Schild, 1959) were constructed. Schild plots were determined from plots of log (CR − 1) versus log [B] according to the equation log (CR − 1) = log [B] − log KB where CR = the ratio of equiactive concentrations of agonist in the presence and absence of antagonist, [B] is the concentration of antagonist, andKB is the equilibrium dissociation constant of antagonist B. pKB = −logKB was calculated assuming a slope of one of the Schild plot.

We were concerned about the possibility of adsorption of peptides and SB 209670 onto components of our organ bath-tissue holder apparatus and therefore experiments were carried out to assess the extent of adsorption. ET-1 (6 nM), together with tracer 125I-ET-1 (40 pM) were added to the organ bath-tissue holder apparatus in the absence of tissue. Samples were taken periodically up to 4 h after addition of ET-1 and counted in a gamma counter (Packard Model B5424). There was a 35 ± 12% (n = 4) loss after 2 h with no further loss after 4 h. In other experiments cumulative concentration-effect curves were established to ET-1 in right atrial trabeculae under conditions to reduce adsorption in which the glassware had been siliconized and 0.05% BSA added to the incubation solution. With trabeculae from the same patient, concentration-effect curves also were constructed in the absence of both siliconized glassware and BSA. There was no difference in ET-1 concentration-effect curves [pEC50 (control, n = 3) − pEC50 (siliconized glassware + BSA,n = 3) = 0.21 ± 0.13 log units,n = 3 hearts]. There was also no difference in pEC50 values for ET-1 in the presence of 10 μM BQ123 with siliconized glassware and BSA, [pEC50(control, n = 5; Fig.1) − pEC50(siliconized glassware + BSA, n = 2) = 0 log units]. Therefore, experiments were carried out in the absence of BSA and without siliconizing glassware. We also determined whether SB 209670 was adsorbed and used 30 nM SB 209670 together with tracer 1 nM [3H]SB 209670. Samples were counted in a liquid scintillation counter (Wallac System 1400). There was no loss of SB 209670 after 4 h.

Statistics.

Comparisons of pEC50 and maximal response values between groups of data were performed by Student's t test (unpaired). Values are expressed as means ± S.E. The significance of differences in the incidence of arrhythmic contractions between β-blocked and non-β-blocked tissues was assessed with the Fisher's exact probability test. Student'st test and Fisher's exact probability test were performed with InStat (GraphPad Software, verson 2.0). P < .05 was used as the limit for statistical significance.

Modulation of contractile force (a) and time to reach 50% relaxation (b) by ET-1. Shown in (a) are the positive inotropic effects of ET-1 in right atrial strips taken from patients undergoing CABG, AVR, or combined CABG/AVR treated with (●) or without (○) β-adrenoceptor antagonists before surgery or right atrial (▪), left atrial (⋄), and right ventricular (▾) trabeculae from hearts in terminal heart failure. Shown in (b) are the effects of ET-1 on the time to reach 50% relaxation in right atrial trabeculae from patients undergoing CABG (●) and right ventricular trabeculae from explanted hearts (▾). Positive inotropic effects were expressed as a percentage of the response obtained to 9.25 mM Ca2+. Values shown are means ± S.E. (vertical lines) where larger than symbol size (n = 3–28 patients).

ET-1 caused positive inotropic effects in cardiac tissues taken from explanted hearts with terminal heart failure. The responses to ET-1 in right atrial trabeculae from these hearts were not different to those obtained in right atrium from coronary artery bypass grafting procedures, i.e., hearts not in failure (P > .05; Fig.2; Table 2). ET-1 also caused concentration-dependent positive inotropic effects in 9 of 12 human left atrial trabeculae from three explanted hearts (Fig. 2). The maximal positive inotropic effect of ET-1 in left atrium was less than in right atrium (P < .05; Fig. 2; Table 2), however, the potency of ET-1 was similar (P > .05; Table 2). ET-1 caused positive inotropic effects in 7 of 15 human right ventricular trabeculae from nine patients. In trabeculae that responded to ET-1, the maximal positive inotropic effect was less than in right atrium (P < .05; Fig. 2; Table 2), however, the potency of ET-1 was similar (P > .05; Table 2).

Effects of ET-1 on Time Course of Contraction.

ET-1 caused a concentration-dependent prolongation of the time to reach 50% relaxation (t50%) in atrium (Fig. 2). In right ventricular trabeculae from explanted hearts, the cumulative addition of ET-1 caused no change int50% (Fig. 2).

Positive Inotropic Effects of ET-2, ET-3, and Sarafotoxin S6c in Human Right Atrium.

ET-2, ET-3, and sarafotoxin S6c caused concentration-dependent positive inotropic effects in human right atrium (Fig. 3). The isoforms of ET-1, ET-2, and ET-3 had similar potencies and caused similar maximal positive inotropic effects (Table 3); however, it was noticeable that in comparision to ET-1 and ET-2, ET-3 caused smaller effects at concentrations up to 6 nM (Fig. 3). Sarafotoxin S6c was more potent than the ET isoforms but caused a smaller maximal positive inotropic effect (Fig. 3; Table 3).

Comparison of positive inotropic effects of ET-1 (○), ET-2 (▴), ET-3 (▪), and sarafotoxin S6c (■) in human right atrium from patients undergoing CABG. Positive inotropic effects were expressed as a percentage of the response obtained by raising the Ca2+ concentration to 9.25 mM at the end of the experiment. Values shown are mean ± S.E. (vertical lines) where larger than symbol size (n = 6–53 patients).

Antagonism of the positive inotropic effects of ET-1 (a) and sarafotoxin S6c (b) by SB 209670 in right atrium from nonfailing hearts. Shown are concentration-effect curves to agonists in the absence (○) or presence of 30 nM (●), 100 nM (▪), 300 nM (▾), 1 μM (♦), or 3 μM (▴) SB 209670. A Schild plot (c) was constructed for SB 209670 with 100 nM, 300 nM, and 1 μM for experiments with ET-1 (●), which gave a slope value of 0.80 ± 0.10 with a pKB value of 7.0 ± 0.1. The higher concentration, 3 μM SB 209670, was not used in the pKB calculation because it caused nonlinearity of the Schild plot (dotted line and ○) and was not completely surmounted by 2 μM ET-1, the highest concentration used. In (a), the ET-1 concentration-effect curve in the absence of SB 209670 is a mean control curve. Concentration-ratios used for the Schild-plot (c) were obtained from individual experiments. A Schild plot also was constructed for SB 209670 against sarafotoxin S6c (▪) with slope 0.94 ± 0.07 and pKB value of 7.9 ± 0.1. The mean sarafotoxin S6c concentration-effect curve in the absence of SB 209670 is a mean control curve. Concentration-ratios used for the Schild-plot were obtained from individual experiments. Positive inotropic effects were expressed as a percentage of the response obtained to 9.25 mM Ca2+. Values shown are means ± S.E. (vertical lines) where larger than symbol size (n = 5–17 individual experiments).

SB 209670 also caused concentration-dependent rightward shifts of the sarafotoxin S6c concentration-effect curve (Fig. 4). The slope of the Schild plot was 0.94 ± 0.07, which was not significantly different to unity. A pKB value of 7.9 ± 0.1, n = 17 patients was calculated.

Lack of antagonism by sarafotoxin S6c against the positive inotropic effects of ET-1 in right atrium from nonfailing hearts. Shown are concentration-effect curves to ET-1 in the absence (○) or presence of 200 nM sarafotoxin S6c (▪). Positive inotropic effects were expressed as a percentage of the response obtained to 9.25 mM Ca2+. Values shown are means ± S.E. (vertical lines) (n = 4 individual experiments).

Blockade of Effects of ET-1 by BQ123 and A-127722 in Human Coronary Artery.

In view of the small blocking effect of BQ123 and A-127722 in right atrium, we tested their ability to block ET-1-mediated contraction of human coronary arteries from one patient with identical tissue bath equipment as for right atrium. ET-1 caused concentration-dependent increases in contractile force that were blocked by 10 μM BQ123 (pKB 6.50) and 1 μM A-127722 (pKB 7.63) (Fig.6).

Arrhythmogenic Effects of ET-1 and Sarafotoxin S6c in Right Atrium from Nonfailing Hearts.

ET-1 (20 nM) caused arrhythmic contractions in right atrial trabeculae that were consistently prevented (13/13 trabeculae from 10 patients) by preincubation of tissues with 10 μM SB 209670 (Fig. 7) but not by 10 μM BQ123 (four trabeculae from three patients) or 1 μM A-127722 (four trabeculae from four patients) (Fig.8). Sarafotoxin S6c (20 nM) also caused arrhythmic contractions that were prevented (7/7 trabeculae from 4 patients) by preincubation with 10 μM SB 209670 (Fig.9). ET-1-induced arrhythmic contractions in atria from non-β-blocker-treated and β-blocker-treated patients were concentration and pacing-frequency dependent (Fig.10). The incidence of ET-1-induced arrhythmic contractions in tissues taken from patients pretreated with or without β-adrenoceptor antagonists before surgery was investigated further with 6 nM ET-1. There was a higher incidence of ET-1-induced arrhythmic contractions in tissues taken from patients pretreated with β-adrenoceptor antagonists at all pacing rates except at 2 Hz than in atria from non-β-blocker-treated patients (Fig.11). We also investigated whether spontaneous contractions could be induced by 100 nM ET-1 in nonstimulated right atrial tissue. Trabeculae were set up and set at 50% Lmax and the stimulator turned off. Spontaneous contractions were observed in seven of nine trabeculae from three patients undergoing coronary artery bypass surgery (Fig. 11). Verapamil (100 nM) reduced, but did not completely reverse, spontaneous contractions in four of four trabeculae from two patients (Fig.12).

ET-1 (20 nM) -evoked arrhythmic contractions and prevention by 10 μM SB 209670. Shown are two atrial strips from a 72-year-old female patient undergoing CABG treated with frusemide, glyceryl trinitrate, simvastatin, lisinopril, amiodarone, and prednisolone before surgery. Top and bottom strips were treated identically except that the bottom strip was preincubated with 10 μM SB 209670 for 60 min before commencement of the experimental protocol. A forward staircase (0.1–2 Hz) was run (a) followed by a backward staircase (2–0.1 Hz) with 2-min intervals between changes in frequency (b). ET-1 was added to both tissues (arrows) (c) and after equilibration another backward staircase with 2-min intervals was run (d). ET-1 induced arrhythmic contractions in the upper strip were not observed in the presence of SB 209670 (bottom strip). There were no arrhythmic contractions in another time-matched strip that was not treated with ET-1 or SB 209670 (data not shown). Solid bars indicate 2-min periods in which the stimulator was turned off.

Persistence of ET-1-induced arrhythmic contractions in the presence of ETA receptor blockade with BQ123 or A-127722. Shown are four right arial strips from a 70-year-old female patient undergoing coronary artery bypass surgery treated with amlodipine, glyceryl trinitrate, isosorbide mononitrate, and atenolol. Tissue (b) was incubated with 10 μM BQ123 and tissue (c) was incubated with 1 μM A-127722. Forward (0.1–2 Hz) and backward staircases (2–0.1 Hz) were carried out as in Fig. 7 during which time no arrhythmic contractions were observed (data not shown). Shown is the reverse staircase (2–0.1 Hz) with 2-min intervals between changes in frequency of stimulation following equilibration with 20 nM ET-1 (tissues a–c) in the absence (a) or presence of BQ123 (b) or A-127722 (c). Tissue (d) was a time-matched control. ET-1-induced arrhythmic contractions were maintained in the presence of BQ123 or A-127722. Solid bars show time periods when the stimulator was turned off.

Sarafotoxin S6c-evoked arrhythmic contractions and prevention by SB 209670. Shown are two atrial strips from a 78-year-old male patient undergoing CABG treated with atenolol, amlodipine, quinapril, and isosorbide mononitrate before surgery. Top and bottom strips were treated identically except that the bottom strip was preincubated with 10 μM SB 209670 for 60 min before commencement of the experimental protocol. The experiment was carried out as in Fig. 7with forward and backward staircases in the absence of sarafotoxin S6c (data not shown). Shown is the backward staircase in the presence of sarafotoxin S6c (arrows) with 2-min intervals between changes in frequency (solid bars) when the stimulator was turned off. SB 209670 (10 μM) prevented 20 nM Sarafoxin S6c-induced arrhythmic contractions.

Incidence of arrhythmic contractions induced by 6 nM ET-1 in atrial tissues taken from patients undergoing CABG surgery. Open columns show the incidence in tissues taken from patients not receiving β-adrenoceptor antagonists (23 strips from 12 patients); open plus closed columns show the incidence in tissues from patients receiving β-adrenoceptor antagonists before surgery (35 strips from 19 patients). Values beneath the columns indicate pacing frequency (Hz) immediately before the rest period in which arrhythmic contractions were observed. Values above the columns are P values obtained with the Fisher's exact probability test to compare the incidence of arrhythmic contractions in β-blocked and non-β-blocked tissues.

ET-1-induced arrhythmic contractions in a quiescent atrial trabeculum taken from a 63-year-old male patient undergoing CABG. Drug therapy before surgery included atenolol, enalapril, simvastatin, and glyceryl trinitrate. The trabeculum was set up at 50% Lmax and the stimulator turned off. ET-1 (100 nM) was added at time zero min, first arrow. Spontaneous contractions appeared after a 7-min ET-1 incubation. Verapamil (100 nM) was added (second arrow, time 60 min), which reduced, but did not abolish spontaneous contractions that were still evident at time 120 min.

Discussion

This study addresses mainly the question of which ET receptors mediate the positive inotropic effects of several agonists in human atrial tissues. We also show that ET-1 causes arrhythmic contractions in right atrial tissues. Only a minor component of the atrial positive inotropic effects of ET-1 was mediated through ETA receptors but mostly through non-ETA and non-ETBreceptors. Sarafotoxin S6c elicited positive inotropic effects in atrium through a second distinct receptor population. The ET-1-evoked arrhythmias were not mediated through ETAreceptors.

Characteristics of Positive Inotropic Effect of ET-1.

ET-1 caused concentration-dependent positive inotropic effects in human cardiac tissues with the maximal effect in right atrium > left atrium = right ventricle. Both Davenport et al. (1989) and Moravec et al. (1989) showed that ET-1 was less effective at causing increases in contractile force in human right ventricle compared with right atrium. Davenport et al. (1989) also reported right ventricular trabeculae that did not respond to ET-1. Interestingly, there was little or no evidence of regulation of the mechanisms responsible for causing positive inotropic effects in human right atrium because ET-1 had similar potencies and maximal effects in atria from patients chronically treated with or without β-adrenoceptor antagonists and patients with terminal heart failure. This is unlike the responses caused by stimulation of some human right atrial Gs protein-coupled receptors, β2-adrenoceptors (Hall et al., 1990), 5-HT4- (Sanders et al., 1995), and H2-receptors (Sanders et al., 1996), and to a minor extent β1-adrenoceptors (Molenaar et al., 1997) that are sensitized in atrial tissues taken from patients treated with β-adrenoceptor antagonists. Furthermore, maintenance of positive inotropic effects of ET-1 in terminal heart failure is unlike β2-adrenoceptor-mediated responses for (−)-epinephrine (in the presence of 300 nM CGP 20712A) in human right atrium (pEC50 heart failure 6.7 ± 0.2, n = 6; coronary artery bypass grafting 7.2 ± 0.1, n = 12; P = .01; unpublished data) and β1-adrenoceptor responses in ventricle (Bristow et al., 1986).

The positive inotropic effects of ET-1 in human right atrium were associated with a prolongation of the time to reach 50% relaxation (t50%) as seen by others (Meyer et al. 1996). In right ventricle from hearts with terminal heart failure, there was no change in t50%, at variance with a prolongation of t50reported by Pieske et al. (1999) for human left ventricular strips. Our results show important differences between the mechanisms by which ET-1 and β-adrenoceptor agonists mediate changes in contractile force. We have shown that selective stimulation of β1- or β2-adrenoceptors causes positive inotropic effects and hastening of relaxation that is associated with cAMP-dependent protein kinase phosphorylation of phospholamban and troponin I in human right atrial and right ventricular trabeculae (Kaumann and Molenaar, 1997; Kaumann et al., 1999). Prolongation of relaxation caused by ET-1 suggests that these pathways are not activated by ET-1 in human atrium.

ET Receptor Heterogeneity: Minor Role of ETAReceptors.

Several ET receptor antagonists were used to characterize the receptors responsible for mediating the cardiostimulant effects of ET-1 and sarafotoxin S6c in right atria from nonfailing hearts. In previous functional studies (Clozel et al., 1994), the nonpeptide antagonist bosentan (formally Ro 47-0203) was reported to competitively antagonize the effects of ET-1 in rat aorta with a pA2 value of 7.3, rabbit superior mesenteric artery (ETB, pA2= 6.7) and rat trachea (ETB, pA2 = 5.9). A concentration of bosentan (10 μM) that would have been expected to block the effects of ET-1 in human right atrium if the receptors were identical with those described in the study of Clozel et al. (1994) was ineffective. Furthermore, it has recently been shown that 3 μM bosentan causes a 1 log rightward shift of the concentration-positive effect curve to ET-1 in human left ventricular strips (Pieske et al. 1999), suggesting that human atrial ET receptors differ from human ventricular receptors. We observed only a ½ log rightward shift of the ET-1 concentration-effect curve with 100 μM bosentan. Because bosentan is a relatively nonselective blocker of ETA and ETBreceptors it appears that the human atrial receptors that mediate positive inotropic effects of ET-1 are mostly neither of ETA nor ETB nature.

Little involvement of ETA receptors also was seen with ETA-selective blockers. High concentrations of the ETA selective antagonists BQ123 (Ihara et al., 1991) (10 μM) and A-127722 (Opgenorth et al., 1996) (1 μM) only caused minor shifts of the lower portion of the ET-1 concentration-effect curve. BQ123 has been reported to block ET-1-induced contractile effects with affinity (pA2) values of 7.4 in porcine coronary artery (Ihara et al., 1991), (pKB) 7.8 in rat aorta (Ohlstein et al., 1994a), and (apparent pA2) 6.4 to 6.8 in proximal human coronary arteries (Godfraind, 1993), including this study (pKB) 6.5. A-127722 is a high-affinity selective ETA receptor antagonist with an affinity (pA2) of 9.2 against ET-1-induced contractile effects in rat aorta (Opgenorth et al., 1996). In human coronary arteries, it blocked ET-1-mediated contraction with lower affinity (pKB = 7.6, present study). Our studies with BQ123 and A-127722 in coronary arteries and cardiac muscle were carried out with identical tissue bath equipment. The agreement between the affinity of BQ123 for ETAreceptors in human coronary arteries determined in our study (pKB = 6.5) and others (apparent pA2 = 6.4–6.8, Godfraind, 1993; pKB = 5.75, Bax et al., 1994; pKB = 7.0, Maguire and Davenport, 1995), together with the greater ability of BQ123 and A-127722 to block the contractile effects of ET-1 in coronary arteries compared with right atrium clearly show that adsorption of the antagonists to components of the tissue bath apparatus cannot explain their failure to block the effects of ET-1 in right atrium. If ETAreceptors were mediating the atrial effects of ET-1 one would expect at least a 1½ log unit rightward shift of concentration-effect curves in the presence of 10 μM BQ123 or 1 μM A-127722, but there was only a ½ log partial shift with either blocker, in agreement with the argument that ETA receptors only play a minor role in the mediation of the positive inotropic effects of ET-1 and only at concentrations of ET-1< 6 nM.

Our conclusion for only a small mediation of ET-1 effects through ETA differs from results of Meyer et al. (1996), who reported that the positive inotropic response to 100 nM ET-1 is markedly reduced by 200 nM BQ123. We tried to repeat the experiment ofMeyer et al. (1996) but for unknown reasons failed to detect any blockade of the effects of 100 nM ET-1 by 200 nM BQ123 (five atrial trabeculae from four patients; K.M.B., unpublished data).

Interestingly, Pieske et al. (1999) reported marked blockade by low BQ123 concentrations (30–300 nM) of the positive inotropic effects of ET-1 in human left ventricular strips, suggesting that the ET-1 effects in this cardiac region are mediated through ETAreceptors. As argued above with bosentan, the marked differences between atrial and ventricular blockade of ET-1 effects by BQ123 is consistent with the concept that most atrial ET-1 receptors differ from ventricular ETA receptors.

SB 209670 also competitively blocked the positive inotropic effects of sarafotoxin S6c in human right atrium with a pKB value of 7.9. This value was higher than that obtained against ET-1 (7.0), indicating ET-1 (at least at low concentrations, see Fig. 4c) and sarafotoxin S6c cause cardiostimulant effects in human right atrium through different receptors. This was confirmed by experiments in which it was shown that 200 nM sarafotoxin S6c only caused a 0.2 log unit rightward shift (not significant) of the concentration-effect curve to ET-1 and no further increase with 1 μM sarafotoxin S6c. High (micromolar) concentrations of ET-1 may however stimulate the sarafotoxin S6c receptor. The concentration-effect curve to ET-1 in the presence of 3 μM SB 209670 was shallow, suggesting stimulation of multiple receptors and which caused a tendency to convergence of the Schild plots for SB 209670 with ET-1 and sarafotoxin S6c. In contrast to human atrium, human ventricle does not appear to respond with positive inotropic responses to sarafotoxin S6c (Pieske et al., 1999), adding a further difference between human atrial and ventricular ET receptors.

The study of Douglas et al. (1995) also reported the existence of a mammalian putative ETC receptor that mediates 10 μM BQ123-insensitive contractile effects to ET receptor agonists in rabbit saphenous vein. In their study, SB 209670 was equally effective in blocking the contractile effects of ET-1 and sarafotoxin S6c and it had similar affinity to bosentan against the agonist effects of sarafotoxin S6c. Both ET-3 and sarafotoxin S6c were more potent than ET-1 for contractile effects. The differences with our data indicate the lack of involvement of a putative ETCreceptor in human atrium.

Our studies show that ET-1 causes minor cardiostimulant effects in human right atrium by stimulating an ETA receptor at low concentrations and most effects through another unclassified receptor at higher concentrations. Sarafotoxin S6c causes positive inotropic effects by stimulation of a receptor that is pharmacologically distinct from receptors activated by ET-1. However, there is no evidence from recombinant ET receptors for the presence of “non-ETA, non-ETB ” receptors in human heart at the present time. Therefore it may be necessary to consider an alternative hypothesis, that the “non-ETA, non-ETB”-mediated effects of ET-1 and sarafotoxin S6c may be due to stimulation of conformations of the cloned ETA or ETB receptors that have low affinity for selective antagonists. Conformational differences have been proposed for other receptors (β1- and β2-adrenoceptors) to explain differences in the potency of (−)-CGP 12177 for inotropic effects, arrhythmic effects, and antagonism (Pak and Fishman, 1996; Freestone et al., 1999; Lowe et al., 1999).

Arrhythmic Contractions Induced by ET-1.

ET-1 and sarafotoxin S6c caused frequency-dependent arrhythmic contractions in our model of human right atrium and also in nonstimulated right atrium. Arrhythmic contractions were prevented by the ET receptor antagonist SB 209670.

There was a higher incidence of ET-1-induced arrhythmic contractions in tissues obtained from patients treated with β-adrenoceptor antagonists before coronary artery bypass surgery. This trend has previously been observed for Gs protein-coupled receptors, β1-, β2-adrenoceptor (Kaumann and Sanders, 1993), 5-HT4- (Kaumann and Sanders, 1994), and H2-receptor (Sanders et al., 1996)-mediated arrhythmic contractions but not as yet for receptors coupled to other second messenger systems. The higher incidence of arrhythmic contractions observed for ET-1 in tissues obtained from patients treated with β-adrenoceptor antagonists may suggest a general increased susceptibility for arrhythmic contractions to a variety of arrhythmogenic agents. Atenolol and metoprolol were the β-adrenoceptor antagonists prescribed for patients undergoing open-chested heart surgery from which right atrium was obtained for arrhythmia studies. The increased general susceptibility to arrhythmias in atria from β-blocker-treated patients could be due to a reduction in Giα protein levels, as found by Sigmund et al. (1996) in patients with dilated and ischemic cardiomyopathy chronically treated with metropolol. In this context, Eschenhagen (1996) has suggested that Gi proteins may have a protective role by preventing the production of arrhythmias.

It has been reported that in human atrial homogenates, ETA receptors mediate formation of inositol phosphates but it is uncertain whether this occurred in atrial myocytes (Pönicke et al., 1998) and is irrelevant to the ability of ET-1 to enhance human atrial contractile force and elicit arrhythmias because these effects are mostly not mediated through ETA receptors. Burrell et al. (1999) also found alkalinization of the cytoplasm by ∼0.25 pH units before the onset of the ET-1-evoked increase in Ca2+ amplitude, suggesting an increase in Na+/H+ exchange activity, as expected from activation of this target of protein kinase C (Krämer et al., 1991; Meyer et al., 1996). These data and consequent mechanisms may explain why verapamil, which blocks mainly L-type Ca2+ channels, failed to abolish ET-1-evoked arrhythmias.

Possible Clinical Relevance.

It has been shown and argued that atenolol is likely to be washed out of cardiac tissues in our protocol (Hall et al., 1990), as is metoprolol (A.J.K. and P.M., unpublished data). Therefore, the ET-1-evoked arrhythmias in our tissues obtained from patients treated with β-adrenoceptor antagonists may be clinically relevant to the β-adrenoceptor blockade withdrawal syndrome (Prichard et al., 1983). It is possible that ET-1 may contribute to transient postcardiac surgical supraventricular arrhythmias together with other arrhythmic agents. Plasma levels of ET-1 are increased as a result of open-chested cardiac surgery (Knothe et al., 1996; Te Velthuis et al., 1996) and remain high for at least 1 day postoperatively (Knothe et al., 1996). This may be relevant in terms of the onset of atrial fibrillation that can occur on the day of surgery, but the peak incidence is 2 days after surgery (Fuller et al., 1989; Kalman et al., 1995). Although plasma levels of ET-1 are not high enough to directly stimulate human cardiac muscle, it would be more likely that locally synthesized and abluminally released ET-1 (Wagner et al., 1992) would directly stimulate cardiac muscle. It remains to be determined whether cardiac ET receptor antagonists will be of value for this disorder. It is interesting to note that SB 209670 prevented exogenous ET-1-induced fatal ventricular arrhythmias in an in vivo canine model, suggesting a broader therapeutic spectrum of antiarrhythmic activity (Douglas et al., 1998).

Conclusions.

The following conclusions can be drawn from the present study. First, ET-1 increases contractile force in both human atrium and ventricle. Sarafotoxin S6c causes positive inotropic effects in atrium but not in ventricle. Most atrial ET receptors activated by ET-1 are of non-ETA nature and differ from atrial receptors activated by sarafotoxin S6c. Second, ventricular ET receptors are different from atrial receptors. Third, ET-1 and sarafotoxin S6c mediate pacing frequency-dependent atrial arrhythmias that are prevented by SB 209670. And fourth, ET-1-induced arrhythmias were mediated through non-ETA receptors.

Acknowledgments

We thank the cardiac surgeons of the Royal Melbourne Public and Private Hospitals, the Alfred Hospital and The Prince Charles Hospital who carefully provided cardiac samples, and the many theater staff who coordinated collection of tissues. P.M. wishes to thank Debbie Beirne at The Prince Charles Hospital for assistance.

↵1 This work was supported by the National Heart Foundation of Australia (K.M.B., postgraduate scholarship), the National Health and Medical Research Council of Australia (P.M.), and British Heart Foundation (A.J.K.).